Measurement of cardiac output and blood volume by non-invasive detection of indicator dilution

ABSTRACT

A system for evaluating the cardiovascular system parameters using indicator dilution and non-invasive or minimally invasive detection and calibration methods are disclosed. Intravascular indicators are stimulated, and emissions patterns detected for computation of cardiac output, cardiac index, blood volume and other indicators of cardiovascular health.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application claims priority to and incorporates by referenceearlier-filed U.S. Provisional Patent Application Ser. No. 60/760,548filed Jan. 20, 2006 entitled “Measurement of Cardiac Output and BloodVolume by Non-Invasive Detection of Indicator Dilution,”. Thisapplication is also a continuation-in-part of U.S. patent applicationSer. No. 10/847,480, filed May 17, 2004, entitled “Measurement ofCardiac Output and Blood Volume by Non-Invasive Detection of IndicatorDilution,” now U.S. Pat. No. 7,590,437 which is a continuation of U.S.patent application Ser. No. 10/153,387, filed May 21, 2002 (now U.S.Pat. No. 6,757,554, issued Jun. 29, 2004) entitled “Measurement ofCardiac Output and Blood Volume by Non-Invasive Detection of IndicatorDilution,” which claims priority to U.S. Provisional Application No.60/292,580 filed on May 22, 2001. This application also relates to U.S.Patent Application Ser. No. 60/747,464 filed May 17, 2006 entitled“Method and Apparatus for Measurement of Cardiac Output and Blood Volumeby Non-Invasive Detection of Indicator Dilution for Hemodyalisis,”; andU.S. Provisional Patent Application No. 60/747,401 filed May 16, 2006entitled “Method for Dye Injection for the Transcutaneous Measurement ofCardiac Output,”. The content of all of these applications isincorporated herein by reference.

FIELD OF THE INVENTION

This invention pertains to the detection of parameters of cardiovascularsystem of a subject.

GENERAL BACKGROUND AND STATE OF THE ART

Cardiac output is a central part of the hemodynamic assessment inpatients having heart disease, acute hemodynamic compromise orundergoing cardiac surgery, for example. Cardiac output is a measure ofthe heart's effectiveness at circulating blood throughout thecirculatory system. Specifically, cardiac output (measured in L/min) isthe volume of blood expelled by the heart per beat (stroke volume)multiplied by the heart rate. An abnormal cardiac output is at least oneindicator of cardiovascular disease.

The current standard method for measuring cardiac output is thethermodilution technique (Darovic, G. O. Hemodynamic monitoring:invasive and noninvasive clinical application. 2nd Ed. W.B. Saunders,1995). Generally, the technique involves injecting a thermal indicator(cold or heat) into the right side of the heart and detecting a changein temperature caused as the indicator flows into the pulmonary artery.

Typically, the thermodilution technique involves inserting aflow-directed balloon catheter (such as a Swan-Ganz catheter) into acentral vein (basilic, internal jugular or subclavian) and guiding itthrough the right atrium and ventricle to the pulmonary artery. Theballoon catheter is typically equipped with a thermistor near its tipfor detecting changes in blood temperature. A rapid injection of a bolusof chilled glucose solution (through a port in the catheter located inthe vena cava near the right atrium) results in a temperature change inthe pulmonary artery detected with the thermistor. The measuredtemperature change is analyzed with an external electronic device tocompute the cardiac output. The algorithm implemented in thiscomputation is typically a variant of the Stewart-Hamilton technique andis based on the theory of indicator mixing in stirred flowing media(Geddes L A, Cardiovascular devices and measurements. John Wiley & Sons.1984).

Thermodilution measurements of cardiac output are disadvantageous forseveral reasons. First, placement of the thermodilution balloon catheteris an expensive and invasive technique requiring a sterile surgicalfield. Second, the catheter left in place has severe risks to thepatient such as local infections, septicemia, bleeding, embolization,catheter-induced damage of the carotid, subclavian and pulmonaryarteries, catheter retention, pneumothorax, dysrrhythmias includingventricular fibrillation, perforation of the atrium or ventricle,tamponade, damage to the tricuspid values, knotting of the catheter,catheter transection and endocarditis. Third, only specially trainedphysicians can insert the balloon catheter for thermodilution cardiacoutput. Last, thermodilution measurements of the cardiac output are tooinvasive to be performed in small children and infants.

Another method used for measuring cardiac output is the dye indicatordilution technique. In this technique, a known volume and concentrationof indicator is injected into the circulatory flow. At a downstreampoint, a blood sample is removed and the concentration of the indicatordetermined. The indicator concentration typically peaks rapidly due tofirst pass mixing of the indicator and then decreases rapidly as mixingproceeds in the total blood volume (˜10 seconds; first passconcentration curve). Further, indicator concentration slowly diminishesas the indicator is metabolized and removed from the circulatory systemby the liver and/or kidneys (time depending upon the indicator used).Thus, a concentration curve can be developed reflecting theconcentration of the indicator over time. The theory of indicatordilution predicts that the area under the first pass concentration curveis inversely proportional to the cardiac output.

Historically, indicator dilution techniques have involved injecting abolus of inert dye (such as indocyanine green) into a vein and removingblood samples to detect the concentration of dye in the blood over time.For example, blood samples are withdrawn from a peripheral artery at aconstant rate with a pump. The blood samples are passed into an opticalsensing cell in which the concentration of dye in the blood is measured.The measurement of dye concentration is based on changes in opticalabsorbance of the blood sample at several wavelengths.

Dye-dilution measurements of cardiac output have been found to bedisadvantageous for several reasons. First, arterial blood withdrawal istime consuming, labor intensive and depletes the patient of valuableblood. Second, the instruments used to measure dye concentrations(densitometer) must be calibrated with samples of the patient's ownblood containing known concentrations of the dye. This calibrationprocess can be very laborious and time consuming in the context of thelaboratory where several samples must be run on a daily basis. Further,technical difficulties arise in extracting the dye concentration fromthe optical absorbance measurements of the blood samples.

A variation on the dye-dilution technique is implemented in the NihonKohden pulse dye densitometer. In this technique, blood absorbancechanges are detected through the skin with an optical probe using avariation of pulse oximetry principles. This variation improves on theprior technique by eliminating the necessity for repeated bloodwithdrawal. However, as described above, this technique remains limitedby the difficulty of separating absorbance changes due to the dyeconcentration changes from absorbance changes due to changes in bloodoxygen saturation or blood content in the volume of tissue interrogatedby the optical probe. This method is also expensive in requiring largeamounts of dye to create noticeable changes in absorbance and a lightsource producing two different wavelengths of light for measuring lightabsorption by the dye and hemoglobin differentially. Even so, the highbackground levels of absorption in the circulatory system makes thistechnique inaccurate. Finally, where repeat measurements are desired,long intervals must ensue for the high levels of the indicator to clearfrom the blood stream. Thus, this technique is inconvenient for patientsundergoing testing and practitioners awaiting results to begin or altertreatment.

Other approaches for measuring cardiac output exist which are not basedon indicator dilution principles. These include ultrasound Doppler,ultrasound imaging, the Fick principle applied to oxygen consumption orcarbon dioxide production and electric impedance plethysmography(Darovic, supra). However, these techniques have specific limitations.For instance, the ultrasound techniques (Doppler and imaging) requireassumptions on the three-dimensional shape of the imaged structures toproduce cardiac output values from velocity or dimension measurements.

Blood volume measures the amount of blood present in the cardiovascularsystem. Blood volume is also a diagnostic measure that is relevant toassessing the health of a patient. In many situations, such as during orafter surgery, traumatic accident or in disease states, it is desirableto restore a patient's blood volume to normal as quickly as possible.Blood volume has typically been measured indirectly by evaluatingmultiple parameters (such as blood pressure, hematocrit, etc.). However,these measures are not as accurate or reliable as direct methods ofmeasuring blood volume.

Blood volume has been directly measured using indicator dilutiontechniques (Geddes, supra). Briefly, a known amount of an indicator isinjected into the circulatory system. After injection, a period of timeis allowed to pass such that the indicator is distributed throughout theblood, but without clearance of the indicator from the body. After theequilibration period, a blood sample is drawn which contains theindicator diluted within the blood. The blood volume can then becalculated by dividing the amount of indicator injected by theconcentration of indicator in the blood sample (for a more detaileddescription see U.S. Pat. No. 6,299,583 incorporated by reference).However, to date, the dilution techniques for determining blood volumeare disadvantageous because they are limited to infrequent measurementdue to the use of indicators that clear slowly from the blood.

Thus, it would be desirable to have a non-invasive, cost effective,accurate and sensitive technique for measuring cardiovascularparameters, such as cardiac output and blood volume.

SUMMARY

The present cardiovascular measurement devices and methods assesscardiovascular parameters within the circulatory system using indicatordilution techniques. Cardiovascular parameters are any measures of thefunction or health of a subjects cardiovascular system.

In one aspect of the present cardiovascular measurement devices andmethods, a non-invasive method for determining cardiovascular parametersis described. In particular, a non-invasive fluorescent dye indicatordilution method is used to evaluate cardiovascular parameters. Themethod may be minimally invasive, requiring only a single peripheral,intravenous line for indicator injection into the circulatory system ofthe patient. Further, a blood draw may not be required for calibrationof the system. Further, cardiovascular parameters may be evaluated bymeasuring physiological parameters relevant to assessing the function ofthe heart and circulatory system. Such parameters include, but are notlimited to cardiac output and blood volume.

Such minimally invasive procedures are advantageous over other methodsof evaluating the cardiovascular system. First, complications andpatient discomfort caused by the procedures are reduced. Second, suchpractical and minimally invasive procedures are within the technicalability of most doctors and nursing staff, thus, specialized training isnot required. Third, these minimally invasive methods may be performedat a patient's bedside or on an outpatient basis. Finally, methods maybe used on a broader patient population, including patients whose lowrisk factors may not justify the use of central arterial measurements ofcardiovascular parameters.

In another aspect of the cardiovascular measurement devices and methods,these methods may be utilized to evaluate the cardiovascular parametersof a patient at a given moment in time, or repeatedly over a selectedperiod of time. The dosages of indicators and other aspects of themethod can be selected such that rapid, serial measurements ofcardiovascular parameters may be made. These methods can be well suitedto monitoring patients having cardiac insufficiency or being exposed topharmacological intervention over time. Further, the non-invasivemethods may be used to evaluate a patient's cardiovascular parameters ina basal state and when the patient is exposed to conditions which mayalter some cardiovascular parameters. Such conditions may include, butare not limited to changes in physical or emotional conditions, exposureto biologically active agents or surgery. For example, embodiments ofthe cardiovascular measurement devices and methods can be used forcardiac output monitoring before, during, or after kidney dialysis;cardiac output monitoring under shock conditions (such as, for example,septic shock, anaphylactic shock, cardiogenic shock, neurogenic shock,hypovolemic shock); cardiac output monitoring during stress tests tobetter understand the heart's ability to increase blood supply to theheart and body while exercising or under other conditions requiringadditional blood flow through the heart; cardiac output monitoringbefore, during, and after chemotherapy treatment to monitor fluidequilibrium in the body; and cardiac output measurements for athletesneeding to understand how their cardiac performance to improve theirathletic performance.

In another aspect of the cardiovascular measurement devices and methods,modifications of the method may be undertaken to improve the measurementof cardiovascular parameters. Such modifications may include alteringthe placement of a photodetector relative to the patient or increasingblood flow to the detection area of the patient's body.

In yet another aspect of the cardiovascular measurement devices andmethods, the non-invasive method of assessing cardiovascular parametersutilizes detection of indicator emission, which is fluorescence, asopposed to indicator absorption. Further, indicator emission may bedetected in a transmission mode and/or reflection mode such that abroader range of patient tissues may serve as detection sites forevaluating cardiovascular parameters, as compared to other methods.Measurement of indicator emission can be more accurate than measurementsobtained by other methods, as indicator emission can be detecteddirectly and independent of the absorption properties of whole blood.

In a further aspect of the cardiovascular measurement devices andmethods, a system for the non-invasive or minimally invasive assessmentof cardiovascular parameters is described. In particular, such a systemmay include an illumination source for exciting the indicator, aphotodetector for sensing emission of electromagnetic radiation from theindicator and a computing system for receiving emission data, trackingdata over time and calculating cardiovascular parameters using the data.

In another aspect of the cardiovascular measurement devices and methods,the methods and system described herein may be used to assesscardiovascular parameters of a variety of subjects. In some embodiments,the methodology can be modified to examine animals or animal models ofcardiovascular disease, such as cardiomyopathies. The methodology of thepresent invention is advantageous for studying animals, such astransgenic rodents whose small size prohibits the use of current methodsusing invasive procedures. The present cardiovascular measurementdevices and methods are also advantageous in not requiring anesthesiawhich can effect cardiac output measurements.

In yet another aspect of the cardiovascular measurement devices andmethods, a noninvasive calibration system can be used to determine theconcentration of circulating indicator dye. In some embodiments, theconcentration of circulating indicator dye can be determined from theratio of emergent fluorescent light to transmitted and/or reflectedexcitation light.

In other embodiments, the methodology can be modified for clinicalapplication to human patients. The present cardiovascular measurementdevices and methods may be used on all human subjects, including adults,juveniles, children and neonates. The present invention is especiallywell suited for application to children, and particularly neonates. Asabove, the present technique is advantageous over other methods at leastin that it is not limited in application by the size constraints of theminiaturized vasculature relative to adult subjects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic depiction of an example of one embodiment ofthe system of the present invention.

FIG. 2 is a fluorescence intensity curve generated using one embodimentof the present invention.

FIG. 3 is a diagrammatic depiction of an example of one embodiment ofthe present invention having a photodetector positioned on the ear skinsurface.

FIG. 4 is a diagrammatic depiction of a user interface of a cardiacoutput computer program useful in conjunction with this invention. Theinterface may depict information regarding values measured and convertedfrom fluorescence to concentration, and parameters of the curve fit forthe values obtained using the method or system.

FIG. 5 is a depiction of a decay of fluorescence intensity curve as afunction of time following injection of a 1 mg dose of indocyanine green(ICG) in an experimental animal.

FIG. 6 is a depiction of a calibration curve for blood ICG concentrationas a function of transcutaneous ICG fluorescence.

FIG. 7 is a depiction of cardiac output and aortic velocity measurementsduring one representative experiment.

FIG. 8 is a depiction of cardiac output measurements derived from siteson the ear surface and on the exposed femoral artery during oneexperiment.

FIG. 9 is a flow chart depicting one method of the present invention.

FIG. 10 illustrates a fluorescence intensity curve that includes anextrapolation that intercepts the point on the curve at which thefluorescence is indicative of the concentration of the indicator whenmixed throughout the volume of blood of the subject.

FIGS. 11A-11D are graphs showing calculated transmission andfluorescence signals at 784 nm and 830 nm for different ICGconcentrations and hemoglobin contents when absorption coefficients arethe same at these two wavelengths.

FIGS. 12A-12D are graphs showing transmission and fluorescence signalsat 784 nm and 830 nm for different ICG concentrations and hemoglobincontents when absorption coefficients vary with wavelength and anadditional absorber is included.

DETAILED DESCRIPTION

The method and system of the present invention are for the evaluation ofcardiovascular parameters of a subject using an indicator dilutiontechnique.

The method of this invention generally involves the injection of aselected amount of indicator into the bloodstream of the subject (FIG.9). Preferably, the indicator is illuminated using a first wavelength ofexcitation light selected to cause the indicator to fluoresce and emit asecond wavelength of light. A photodetector is placed near the subjectfor the detection of the intensity of the emitted second wavelength oflight, which is proportional to the concentration of the indicatorcirculating within the circulatory system. The photodetector transmitsthis intensity information to a computing system, which records andpreferably maps the intensity curve of the indicator detected over time.

Typically, the indicator concentration values increase to a peak rapidlyafter injection of the indicator. Then, the concentration valuesdecrease rapidly, then more steadily as the indicator is mixedthroughout the body circulatory system and metabolized over time. Amicroprocessor driven computation then can calculate from theconcentration curve, the patient's cardiac output and/or blood volumevalues. Additionally, values can be generalized repeatedly using thismethod, at intervals of about every 2-5 minutes.

Indicators. The indicators useful in this invention are preferably inertand biocompatible in that they do not alter cardiovascular parameters,such as heart rate. Further, the indicator is preferably a substancethat once injected, does not diffuse out of the vasculature of thecardiovascular system. Also, the indicator is preferably selected to beone which is metabolized within the body at a rate such that repeatedmeasures using this method may be conducted at intervals of about 2-5minutes. It is also desirable that the background levels of circulatingindicator be cleared between intervals, although measurements may betaken when background levels are not zero. Finally, the indicator can beselected to be detectable by the photodetector system selected.

In one embodiment, a non-invasive dye indicator dilution method may beused to evaluate cardiovascular function. Many different dye indicatorsmay be used within the scope of this invention. Preferably, the dyeindicator is fluorescent having an excitation wavelength and an emissionwavelength in the near infrared spectrum, preferably about 750 nm toabout 1000 nm, and more preferably about 750 nm to about 850 nm.

Most preferably, the indicator used is indocyanine green (ICG; purchasedfor example from Akorn, Decatur or Sigma, St. Louis, Mo.; commercialnames: Diagnogreen©, ICGreen©, Infracyanine©, Pulsion©). ICG has beenpreviously been used to study the microcirculation of the eye, thedigestive system and liver function (Desmettre, T., J. M. Devoisselle,and S. Mordon. Fluorescence properties and metabolic features ofindocyanine green (ICG) as related to angiography. Surv Ophthalmol 45,15-27, 2000). ICG fluoresces intensely when excited at near infraredwavelengths. In the context of this invention, ICG in blood plasma has apeak fluorescence of about 810 to 830 nm with an optimal excitationwavelength of about 780 nm (Hollins, supra; Dorshow, supra). ICG may beadvantageous for use in this invention in remains intravascular becauseit is protein bound. ICG breaks down quickly in aqueous solution, andmetabolites are not fluorescent, minimizing recirculation artifact andreducing the time period between which measurements can be made. Thewavelength of emission of ICG is also within the optical window(750-1000 nm) in which living tissues are relatively transparent tolight.

Other biocompatible fluorescent dyes such as fluorescein and rhodaminewould also be suitable in this invention. Fluorescein in blood plasmahas a peak fluorescence of about 518±10 nm with an optimal excitationwavelength of about 488 nm (Hollins, supra; Dorshow, supra). Rhodaminein blood plasma has a peak fluorescence of about 640±10 nm with anoptimal excitation wavelength of about 510 nm.

Indicator dosage. The dosage of indicator is preferably selected suchthat an amount used is non-toxic to the subject, is present in thecirculatory system for an amount of time adequate to establish anindicator concentration curve, but is metabolized in an amount of timesuch that repeated measurements can be conducted at intervals of about2-5 minutes apart. Further, the indicator is preferably administered tothe subject by injection into a vein.

A dosage of about 0.015 mg/kg is preferable in that this dose leads topeak blood concentrations below 0.002 mg/ml. In this concentrationrange, the measurement of the circulating indicator concentration islinearly related to the intensity of the emission wavelength detected.For example, in a laboratory animal model, about 0.045 mg can beinjected into a 3 kg rabbit (blood volume=200 ml) such that the averagecirculating concentration is about 0.00023 mg/ml whole blood.

Dye dilution techniques have been applied in humans in other methods andsystems using indocyanine green as a dye. Living tissues of humans andanimals are relatively transparent for near infrared wavelengths oflight which allows for transmission of light across several mm of tissueand transcutaneous detection of the fluorescence emission of ICG. Theuse of dosages in the ranges stated above is additionally suitable forhuman use.

Illumination Source. The illumination sources useful in this inventionare preferably selected to produce an excitation wavelength in the nearinfrared spectrum, preferably about 750 nm to about 1000 nm, and morepreferably about 750 to about 850 nm. This selection is advantageous inat least that most tissues are relatively transparent to wavelengths inthis range. Thus, in some embodiments, an indicator in the blood streamis excitable transcutaneously and indicator emissions detectedtranscutaneously. Further, blood constituents do not fluoresce at thesewavelengths, thus there is no other contributor to the measuredfluorescence emission signal. Therefore, this method is advantageous inthat at least the sensitivity of detection in this method is improvedover other methods, which measure indicator absorption, as opposed toemission.

However, it is within the scope of the invention to use otherwavelengths of light, for example in the blue-green or ultraviolet rangeas some tissues are relatively transparent even at these wavelengths.Selection of the illumination source, therefore, can depend in part onthe indicator selected and the tissue from which detection will be made.Preferably, the illumination source is selected to result in the peakemission wavelength of the indicator.

Examples of illumination sources which may be used in this inventioninclude, but are not limited to lamps, light emitting diodes (LEDs),lasers or diode lasers.

In some embodiments, modifications to the system or illumination sourcemay be altered to further to maximize the sensitivity or accuracy of thesystem for measuring indicator concentration. For example, in someembodiments, the excitation wavelength produced by the illuminationsource will be steady. Alternatively, the excitation wavelength producedby the illumination source can be modulated to allow for a lock-indetection technique.

For example, the illumination source may emit light in a periodicvarying pattern having a fixed frequency and the emission recorded bythe photodetector read at the same frequency to improve the accuracy ofthe readings. The periodic varying pattern and frequency can be selectedto improve noise-rejection and should be selected to be compatible withthe rest of the instrumentation (such as the light source andphotodetector).

The illumination source may be adapted to target a detection area of thesubject's tissue from which emission wavelength intensity will berecorded. In some embodiments, the illumination source may comprise anoptic fiber for directing the excitation light to the detection area. Insome embodiments, the illumination source may comprise mirrors, filtersand/or lenses for directing the excitation light to the detection area.

Detection Areas. The target detection area is that location of asubject's tissue which is exposed to the excitation wavelength of lightand/or from which the emission wavelength light intensity output will bemeasured.

Preferably, the method of detection is non-invasive. In theseembodiments, a detection area is selected such that a photodetector canbe placed in proximity to the detection area and emission wavelengthlight intensity measured. Preferably, the photodetector is placedtransdermally to at least one blood vessel, but more preferably a highlyvascularized tissue area. Examples of detection areas include, but arenot limited to fingers, auricles of the ears, nostrils and areas havingnon-keratinized epithelium (such as the nasal mucosa or inner cheek). Inalternative embodiments, the method of detection is minimally invasive.For example, the photodetector can be placed subdermally (within orbeneath the epidermis) and proximate to at least one blood vessel or ina perivascular position.

In yet alternative embodiments, the method of detection is minimallyinvasive. For example, the photodetector can be placed intravascularlyto detect indicator emissions, such as within an artery. In suchembodiments, an external probe for emitting and receiving light may notbe needed. For example, in some embodiments the probe may include afiber optic located within an intravascular catheter. Specifically, thedevice may include an intravascular catheter made of biocompatibleplastic material which contains, embedded in the catheter wall, anoptical fiber 412 that ends at or near the tip 416 of the catheter. Forexample, the catheter may have a diameter of 100 μm or less. The fiberoptic can be used to optically sense the presence and concentration ofendogenous substances in the blood or exogenous substances injected orinfused in the blood stream through the catheter lumen or anothercatheter. A fiber optic connector 414 at the proximal external end ofthe fiber optic connects the fiber to an external monitor. In use, theneedle of an injection syringe can be inserted through the catheterlumen and used to inject the indicator material (meanwhile the cathetermay be allowed to remain within the vein or artery). The injectionneedle may be withdrawn from the catheter after injection. After theindicator has been injected and the indicator has had sufficient time tocirculate through the cardiovascular system, light from a light sourcecan be directed to the blood and circulated indicator via the opticalfiber embedded in the catheter. The optical fiber of the catheter mayalso be used to receive light from the indicator and transmit the lightto the monitor. In alternative embodiments, the catheter may include aplurality of optical fibers for transmitting and/or receiving light usedto obtain measure parameters of interest of the cardiovascular system.Catheters that include optical fibers are described in U.S. Pat. Nos.4,730,622 to Cohen and 5,217,456 to Narciso, the entire contents of eachof which are incorporated by reference. In addition, other sensingdevices and mechanisms may be included in the intravascular probe.

Additionally, the detection area may be arterialized during indicatoremission detection. Examples of conditions resulting in detection areaarterialization include, but are not limited to heating or exposure tobiologically active agents which effect sympathetic system blockade(such as lidocaine).

Photodetector. The detection of indicator emissions can be achieved byoptical methods known in the art. Measurement of indicator concentrationcan be made by administering a detectable amount of a dye indicator andusing a non-invasive, minimally invasive, or intravascular procedures,preferably for continuous detection. The photodetector may be positionedproximately to the detection area of the subject. The photodetector maybe positioned distally or proximately to the site of the illuminationsource.

Fluorescent light is emitted from the indicator with the same intensityfor all directions (isotropy). Consequently, in some embodiments, theemission of the dye can be detected both in “transmission mode” when theexcitation light and the photodetector are on opposite sides of theilluminated tissue and in “reflection mode” when the excitation and thephotodetector are on the same side of the tissue. This is advantageousover other methods at least in that the excitation light and emittedlight can be input and detected from any site on the body surface andnot only optically thin structures.

Photodetectors which are useful in this invention are those selected todetect the quantities and light wavelengths (electromagnetic radiation)emitted from the selected indicator. Photodetectors having sensitivityto various ranges of wavelengths of light are well known in the art.

In some embodiments, modifications to the system are made to furtherenhance the sensitivity or accuracy of the system for measuringindicator concentration. For example in some embodiments, the detectionsystem can incorporate a lock-in detection technique. For example, theexcitation light may be modulated at a specific frequency and a lock-inamplifier can be used to amplify the output of the photodetector only atthat frequency. This feature is advantageous in at least that it furtherimproves the sensitivity of the system by reducing signal to noise andallows detection of very small amounts of fluorescence emission.

In some embodiments a photomultiplier tube is utilized as or operablyconnected with another photodetector to enhance the sensitivity of thesystem. Finally, in some embodiments, additional features, such asfilters, may be utilized to minimize the background of the emissionsignals detected. For example, a filter may be selected whichcorresponds to the peak wavelength range or around the peak wavelengthrange of the indicator emission.

The detected electromagnetic radiation is converted into electricalsignals by a photoelectric transducing device which is integral to orindependent of the photodetector. These electrical signals aretransmitted to a microprocessor which records the intensity of theindicator emissions as correlated to the electrical signal for any onetime point or over time. (For an example of such a device see U.S. Pat.No. 5,766,125, herein incorporated by reference.)

System Calibration.

A) Minimally Invasive Calibration

The method may be minimally invasive in requiring only a singleperipheral blood draw from the circulatory system to be taken forcalibration purposes. In this invention, indicator concentration ispreferably being measured continuously and non-invasively using aphotodetector. However, one blood sample from the subject may bewithdrawn for calibration of the actual levels of circulating indicatorwith the indicator levels detected by the system. For example, a bloodsample may be drawn from the subject at a selected time after theadministration of the indicator into the blood stream. The blood samplemay then be evaluated for the concentration of indicator present bycomparison with a calibration panel of samples having known indicatorconcentrations. Evaluation of the indicator concentration may be madespectrophotometrically or by any other means known in the art. Where thesubject blood concentration of indicator falls within a range of about0.001 to about 0.002 mg/ml, the concentration-fluorescence curve islinear and it crosses the origin of the axes, the fluorescence is zerowhen the concentration is zero. Therefore a single measurement pointsuffices to define the calibration curve, and no further blood samplesneed be taken.

B) Noninvasive Calibration

In another embodiment no blood draw is required for calibration of thissystem. It is noted that the fluorescence of some indicators, such asICG, does not substantially vary from patient to patient and that theskin characteristics are relatively constant for large classes ofpatients. Thus, the fluorescence in the blood of the patient measuredfrom a given site on the body surface can be converted in an absolutemeasurement of ICG concentration, once the curve of indicatorconcentration vs. fluorescence is defined for that site of measurement.

In an exemplary embodiment using noninvasive calibration, theconcentration of a fluorescent indicator (ICG) injected in thebloodstream can be determined without taking a blood sample. A probe(including or connected to one or several photodetectors, as describedabove) can measure the intensity of fluorescent light emitted by the ICGindicator when illuminated by a light source in or near the skin. Theprobe can also measure the intensity of the light from that source thatis reflected by or transmitted through the illumination skin site. Sincethe ratio of emergent fluorescent light to transmitted excitation lightis directly proportional to ICG concentration (see FIGS. 11A-D, FIGS.12A-12D, and Example 3 below), the concentration of ICG can bedetermined from the ratio of emergent fluorescent light to transmittedexcitation light. For example, the graph in FIG. 11C shows that ICGconcentration is directly proportional to the ratio of fluorescent lightto transmitted excitation light. In another example illustrated by thegraph of FIG. 12C, ICG remains directly proportional to the ratio offluorescent light to transmitted excitation light even when factoringthe variations of absorption properties for hemoglobin (Hb) and ICG withwavelength and the absorption by bloodless tissue. While the slopes ofthe lines in FIG. 12C vary slightly depending upon hemoglobin content,the differences between the light ratios are relatively small. Theratios may be normalized by creating a table of coefficients that takeinto account various factors that may affect the light ratios (such asabsorption by bloodless tissue, hemoglobin content, path length, skincolor, moisture on skin surfaces, body hair, and other factors known tothose skilled in the art).

The probe used to transmit and receive light may include a singleoptical fiber, multiple optical fibers for transmitting and/or receivinglight, or other configuration known to those skilled in the art. Theexcitation light that is received and used in the ratio againstfluorescence may be reflected and/or transmitted light. For example, inone embodiment, the light transmitter and receiver can be on the sameskin surface so that the receiver can receive light reflected from thetissue. In such an embodiment, the receiving and transmitting elementare the same optical fiber (See Diamond et al., “Quantification offluorophore concentration in tissue-simulating media by fluorescencemeasurements with a single optical fiber;” Applied Optics, Vol. 42, No.13, May 2003; the contents of which are incorporated herein byreference). In other embodiments, they may be different optical fibers(or other devices known to those skilled in the art). In suchembodiments, the various optical fibers may be spatially positioned inrelation to each other to optimize measurement, as described in Weersinket al. (See Weersink et al., “Noninvasive measurement of fluorophoreconcentration in turbid media with a simple fluorescence/reflectanceratio technique;” Applied Optics, Vol. 40, No. 34, December 2001; andU.S. Pat. No. 6,219,566 to Weersink et al.; the contents of both ofwhich are incorporated herein by reference). In another embodiment, thetransmitter and receiver are positioned substantially opposite eachother to allow transmission of the light (such as forward scattering)from the transmitter, through the tissue, and out of the tissue to thereceiver on the other side of the tissue.

These methods and systems may be utilized to measure severalcardiovascular parameters. Once the system has been calibrated to thesubject (where necessary) and the indicator emissions detected andrecorded over time, the computing system may be used to calculatecardiovascular parameters including cardiac output and blood volume.

Cardiac output calculations. In some embodiments, the cardiac output iscalculated using equations which inversely correlate the area under thefirst pass indicator emission curve (magnitude of intensity curve) withcardiac output. Cardiac output is typically expressed as averages(L/min). The general methods have been previously described (Geddes,supra, herein incorporated by reference).

Classically, the descending limb of the curve is plottedsemi-logrithmically to identify the end of the first pass of indicator.For example, the descending limb of the curve may be extrapolated downto 1% of the maximum height of the curve. The curve can then becompleted by plotting values for times preceding the end time. Finally,the area under this corrected curve is established and divided by thelength (time) to render a mean height. This mean height is converted tomean concentration after calibration of the detector. The narrower thecurve, the higher the cardiac output; the wider the curve, the lower thecardiac output. Several variations of this calculation method are found,including methods that fit a model equation to the ascending anddescending portions of the indicator concentration curve.

Depending upon the indicator type and dosage selected, the curve may notreturn to zero after the end of the first pass due to a residualconcentration of indicator recirculating in the system. Subsequentcalculations of cardiac output from the curve may then account for thisrecirculation artifact by correcting for the background emissions, priorto calculating the area under the curve.

Sequential measurements of a cardiovascular circulatory parameter, suchas cardiac output or blood volume, may be taken. Each measurement may bepreceded by the administration of an indicator to the cardiovascularsystem. Each measurement may be separated by a time period during whichthe indicator that was previously administered is substantiallyeliminated from the circulatory system, for instance by metabolicprocesses.

To obtain a measurement in absolute physical units, e.g., in liters perminute for cardiac output or liters for blood volume, a blood sample maybe taken after each administration of the indicator for calibrationpurposes, as explained in more detail above.

Another approach may be to take a blood sample only after the firstadministration of the indicator and to use this blood sample forcalibration purposes during each subsequent administration of theindicator and measurement of its resulting fluorescence. However, theoperating characteristics of the test equipment may shift during thesetests. The optical properties of the tissue being illuminated may alsochange. The positioning of the illumination source and/or the photodetector may also change. All these changes can introduce errors in thecomputation of the parameter in absolute physical units when thecomputations are based on a blood sample that was taken before thechanges occurred.

These errors may be minimized by measuring the changes that occur afterthe blood sample is taken and by then adjusting the measuredfluorescence intensity to compensate for these measured changes. Thismay be accomplished by measuring the intensity of the illumination lightafter it is transmitted through or reflected by the tissue through whichthe administered indicator passes. This illumination intensitymeasurement may be made shortly before, during or shortly after eachadministration of the indicator. The computations of the cardiovascularparameter that are made during tests subsequent to the first test (whenthe calibrating blood sample was taken) may then be adjusted inaccordance with variations in these illumination intensity measurements.

For example, the computation of the cardiovascular parameter that ismade following the second administration of the indicator may bemultiplied by the ratio of the illumination intensity measurement madeprior to the first administration of the indicator to the illuminationintensity measurement made prior to the second administration of theindicator. If the illumination intensity between the first and secondmeasurements doubles, for example, application of this formula mayresult in a halving of the computation. Other functional relationshipsbetween the measured cardiovascular parameter and the illuminationintensity measurements may also be implemented.

Any equipment may be used to make the illumination intensitymeasurements. In one embodiment, the photo detector that detects thefluorescence intensity may also be used to make the illuminationintensity measurements. The optical filter that removes light at theillumination frequency may be removed during the illumination intensitymeasurements. The leakage of the illumination thought this filter mayinstead be measured and used as the information for the computation.

Another approach to minimizing the number of needed blood samples for asequence of tests is to take advantage of the known relationship betweenthe amount of indicator that is injected, the volume of blood in thecirculatory system and the resulting concentration of the indicator inthat blood.

One step in this approach is to determine the volume of blood in thecardiovascular circulatory system using any technique, such as a tracerdilution technique, applied for instance with the Evans Blue dye. Theconcentration of the indicator after it is administered and mixedthroughout the total blood volume, with no offset for metabolicelimination, may then be computed by dividing the amount of theindicator that is administered by the volume of the blood.

The theoretical magnitude of the intensity of the fluorescence from theindicator after the indicator is mixed throughout the total bloodvolume, without having been metabolized or otherwise eliminated from thecirculatory system, may then be determined from the fluorescence curve.FIG. 10 illustrates one way that this may be done. As shown in FIG. 10,the intensity of the fluorescence of an administered indicator willoften rise quickly after the injection, as illustrated by a sharplyrising portion 1001. The intensity may then decay slowly, as illustratedby a slowly falling portion 1003. A portion of the curve 1004 during theslow decay may be extrapolated until it intercepts a point 1005 on thefast rising portion. The level of the intensity of the fluorescence atthe point 1005 may represent the concentration of the indicator after itis administered and mixed throughout the total blood volume, with nooffset for metabolic elimination, i.e., the concentration of theindicator that was computed above.

Based on this extrapolated point, a conversion factor may then bedetermined that converts the measured intensity of the fluorescence tothe concentration of the indicator in the cardiovascular system. Theconversion factor may be determined by equating it to the ratio of theconcentration of the indicator that was calculated above to themeasurement of the intensity of the fluorescence at the interceptedpoint. The concentration of the indicator at other points on thefluorescence intensity curve shown in FIG. 10 may then be computed bymultiplying the measured fluorescence intensity value by the conversionfactor.

Subsequent administrations of indicator may be made and measured tomonitor the cardiovascular parameter over short or long periods of time.The same computational process as is described above may be used eachtime to determine the absolute physical value of the desiredcardiovascular parameter without having to again take a blood sample.The process may also intrinsically compensate for changes betweenmeasurements, other than changes in blood volume, such as changes in theoperating characteristics of the test equipment, the optical propertiesof the tissue being illuminated, and/or the positioning of theillumination source and/or the photo detector.

All of the foregoing computations, as well as others, may beautomatically performed by a computing system. The computing system mayinclude any type of hardware and/or software.

Results obtained using this system can be normalized for comparisonbetween subjects by expressing cardiac output as a function of weight(CO/body weight (L/min/kg)) or as a function of surface area (cardiacindex=CO/body surface area (L/min/m²)).

Blood volume calculations. In some embodiments, blood volume may bemeasured independently or in addition to the cardiac output. Generalmethods of measuring blood volume are known in the art. In someembodiments, circulating blood volume may be measured using a low doseof indicator which is allowed to mix within the circulatory system for aperiod of time selected for adequate mixing, but inadequate or theindicator to be completely metabolized. The circulating blood volume maythen be calculated by back extrapolating to the instant of injection theslow metabolic disappearance phase of the concentration curve detectedover time (Bloomfield, D. A. Dye curves: The theory and practice ofindicator dilution. University Park Press, 1974). Alternative methods ofcalculation include, but are not limited to those described in U.S. Pat.Nos. 5,999,841, 6,230,035 or 5,776,125, herein incorporated byreference.

This method and system may be used to examine the general cardiovascularhealth of a subject. In one embodiment, the method may be undertaken onetime, such that one cardiac output and or blood volume measurement wouldbe obtained. In other embodiments, the method may be undertaken toobtain repeated or continuous measurements of cardiovascular parametersover time. Further, repeated measures may be taken in conditions wherethe cardiovascular system is challenged such that a subject's basal andchallenged cardiovascular parameters can be compared. Challenges whichmay be utilized to alter the cardiovascular system include, but are notlimited to exercise, treatment with biologically active agent whichalter heart function (such as epinephrine), parasympathetic stimulation(such as vagal stimulation), injection of liquids increasing bloodvolume (such as colloidal plasma substitutes) or exposure to enhancedlevels of respiratory gases.

A schematic of one embodiment of a system 10 useful in the presentinvention is shown in FIG. 1. The system comprises an illuminationsource 12 here a 775 nm laser selected to emit a excitation wavelengthof light 14 which maximally excites ICG, the indicator selected. Herethe illumination source 12 is positioned proximately to the subject 16,such that the excitation wavelength of light 14 is shone transdermallyonto the indicator circulating in the bloodstream. The system alsocomprises a photodetector 20 placed in proximity to the subject's skinsurface 18 for detection of the indicator emission wavelength 22.Optionally, a filter 24 may be used for isolating the peak wavelength atwhich the indicator emits, being about 830 nm. Finally, thephotodetector 20 is operably connected to a microprocessor 26 forstoring the electronic signals transmitted from the photodetector 20over time, and generating the indicator concentration curve (FIG. 2).Optionally, the microprocessor 26 may regulate the illumination sourceto coordinate the excitation and detection of emissions from theindicator, for example using a modulation technique. The microprocessormay also comprise software programs for analyzing the output obtainedfrom the detector 20 such that the information could be converted intovalues of cardiac output or blood volume, for example and/or displayedin the form of a user interface.

In order to demonstrate the utility of the invention, a non-invasiveindicator detection system 10 of the invention was used to repeatedlymonitor cardiac output. With reference to FIG. 1, a fiber optic 12 btransmitted light from illumination source 12 a to the subject's skin18. A second fiber optic 20 b, positioned near the skin 18 transmittedthe emitted light to a photodetector 20. The indicator was intravenouslyinjected. A body portion which included blood vessels near the surfaceof the skin, was irradiated with a laser. A characteristic fluorescenceintensity/concentration curve was obtained upon excitation with laserlight at about 775 nm and detection of the fluorescence at about 830 nm.From this information cardiac output and blood volume for the subjectwas calculated.

The system used for this method may comprise a variety of additionalcomponents for accomplishing the aims of this invention. For example,non-invasive detection is described for monitoring of indicators withinthe circulatory system of the patient. Modifications of the detectors toaccommodate to various regions of the patient's body or to providethermal, electrical or chemical stimulation to the body are envisionedwithin the scope of this invention. Also, calibration of the system maybe automated by a computing system, such that a blood sample is drawnfrom the patient after administration of the indicator, concentrationdetected and compared with known standards and/or the emission curve.Also, software may be used in conjunction with the microprocessor to aidin altering parameters of any of the components of the system oreffectuating the calculations of the cardiovascular parameters beingmeasured. Further, software may be used to display these results to auser by way of a digital display, personal computer or the like.

The utility of the invention is further illustrated by the followingexamples, which are not intended to be limiting.

EXAMPLE 1

Experimental system and method. An implementation of the system andmethod of this invention was tested in rats. The excitation source was a775 nm pulsed diode laser and the fluorescence was detected with adetector being a photomultiplier (PMT) with extended response in thenear-infrared range of the spectrum (FIG. 1). Optic fibers were placedin close contact with the skin of the animal's ear for the excitationand detection of the indicator within the blood stream. After injectionof a 100 □l bolus of ICG (0.0075 mg/ml) into the jugular vein of a rat,the fluorescence intensity trace (indicator concentration recording) wasmeasured transcutaneously at the level of the rat's ear using reflectionmode detection of emissions (FIG. 2).

Calculation of blood volume and cardiac output. The initial rapid riseand rapid decay segments of the fluorescence intensity trace representthe first pass of the fluorescent indicator in the arterial vasculatureof the animal. Such a waveform is characteristic of indicator dilutiontechniques. This portion of the recording is analyzed with one ofseveral known algorithms (i.e. Stewart Hamilton technique) to computethe “area under the curve” of the fluorescence intensity trace whileexcluding the recirculation artifact. Here, the initial portion of thefluorescence trace y(t) was fitted with a model equationy(t)=y₀t^(□)exp(−□t) which approximates both the rising and descendingsegments of the trace. This equation derived from a “tank-in-series”representation of the cardiovascular system has been found fit well theexperimental indicator dilution recordings. The numerical parameters ofthe fit were determined from the approximation procedure, and then the“area under the curve” was computed by numeric integration and used tofind the cardiac output with the known formula:

$Q = {\frac{m}{\int_{0}^{\infty}{{C(t)}{\mathbb{d}t}}} = \frac{{amount}\mspace{14mu}{injected}}{{area}\mspace{14mu}{under}\mspace{14mu}{the}\mspace{14mu}{curve}}}$

Back extrapolation of the slow decay segment of the fluorescenceintensity trace to the instant when ICG is first detected in the blood(time 0) yields the estimated concentration of ICG mixed in the wholecirculating blood volume. By dividing the amount of injected ICG by thisextrapolated ICG concentration at time 0, the circulating blood volumewas computed.

Calibration methods. Indicator concentration C(t) was computed from thefluorescence y(t) using one of two calibration methods. Transcutaneousin vivo fluorescence was calibrated with respect to absolute bloodconcentrations of ICG, using a few blood samples withdrawn from aperipheral artery after bolus dye injection of ICG. The blood sampleswere placed in a fluorescence cell and inserted in a tabletopfluorometer for measurement of their fluorescence emission. Thefluorescence readings were converted into ICG concentrations using astandard calibration curve established by measuring with the tabletopfluorometer the fluorescence of blood samples containing knownconcentrations of ICG.

An alternative calibration procedure which avoids blood loss uses asyringe outfitted with a light excitation—fluorescence detectionassembly. The syringe assembly was calibrated once before the cardiacoutput measurements by measuring ICG fluorescence in the syringe fordifferent concentrations of ICG dye in blood contained in the barrel ofthe syringe. During the measurement of cardiac output, a blood samplewas pulled in the syringe during the slow decay phase of thefluorescence trace, that is the phase during which recirculating dye ishomogeneously mixed in the whole blood volume and is being slowlymetabolized. The fluorescence of that sample was converted toconcentration using the syringe calibration curve and then related tothe transcutaneous fluorescence reading. So long as the ICGconcentrations in blood remain sufficiently low (<0.001 mg/ml), a linearrelationship can be used to relate fluorescence intensity toconcentration.

Either one of these calibration methods can be developed on a referencegroup of subjects to produce a calibration nomogram that would serve forall other subjects with similar physical characteristics (i.e., adults,small children etc.). This is advantageous over prior methods at leastin that an additional independent measurement of the blood hemoglobinconcentration for computation of the light absorption due to hemoglobinis not required.

EXAMPLE 2

A. A Sample Method and System for Measuring Cardiac Output and BloodVolume. Experiments have been performed in New Zealand White rabbits(2.8-3.5 Kg) anesthetized with halothane and artificially ventilatedwith an oxygen-enriched gas mixture (FiO2˜0.4) to achieve a SaO2 above99% and an end-tidal CO2 between 28 and 32 mm Hg (FIG. 4). The leftfemoral artery was cannulated for measurement of the arterial bloodpressure throughout the procedure. A small catheter was positioned inthe left brachial vein to inject the indicator, ICG. Body temperaturewas maintained with a heat lamp.

Excitation of the ICG fluorescence was achieved with a 780 nm laser (LDhead: Microlaser systems SRT-F780S-12) whose output was sinusoidallymodulated at 2.8 KHz by modulation of the diode current at the level ofthe laser diode driver diode (LD Driver: Microlaser Systems CP 200) andoperably connected to a thermoelectric controller (Microlaser Systems:CT15W). The near-infrared light output was forwarded to the animalpreparation with a fiber optic bundle terminated by a waterproofexcitation-detection probe. The fluorescence emitted by the dye in thesubcutaneous vasculature was detected by the probe and directed to a 830nm interferential filter (Optosigma 079-2230) which passed thefluorescence emission at 830±10 nm and rejected the retro-reflectedexcitation light at 780 nm. The fluorescence intensity was measured witha photomultiplier tube (PMT; such as Hamamatsu H7732-10MOD) connected toa lock-in amplifier (Stanford Research SR 510) for phase-sensitivedetection of the fluorescence emission at the reference frequency of themodulated excitation light. The output of the lock-in amplifier wasdisplayed on a digital storage oscilloscope and transferred to acomputer for storage and analysis.

In most experiments, one excitation-detection probe was positioned onthe surface of the ear arterialized by local heating. In some studies,the laser emission beam was separated in two beams with a beam splitterand directed to two measurement sites (ear skin and exposed rightfemoral artery). Two detection systems (PMT+lock in amplifier) were usedfor measurement of the fluorescence dilution traces from the two sites.In all experiments, a complete record of all experimental measurements(one or two fluorescence traces, arterial blood pressure, end-tidal CO2,Doppler flow velocity) was displayed on line and stored for reference.

Calculations. A LabView program was used to control the oscilloscopeused for sampling the fluorescence dilution curves, transfer the datafrom the oscilloscope to a personal computer and analyze the curvesonline for estimation of the cardiac output and circulating bloodvolume. As shown on the program user interface (FIG. 5), the measuredfluorescence dilution trace (a) is converted to ICG blood (b) using thecalibration parameters estimated as described in the next section ofthis application and fitted to a model: C(t)=C₀t^(α)exp(−βt).

The model fit is performed from the time point for which the fluorescentICG is first detected to a point on the decaying portion of the tracethat precedes the appearance of recirculating indicator (identified fromthe characteristic hump after the initial peak in the experimentaltrace). The model equation is used to estimate the “area under thecurve” for the indicator dilution trace. The theory of indicatordilution technique predicts that the area under the concentration curveis inversely proportional to the cardiac output (Q): m/^(∞) ₀∫C(t)dt.

where m is the mass amount of injected indicator and c(t) is theconcentration of indicator in the arterial blood at time t. The programalso fits the slow decaying phase of the measurement to a singleexponential to derive the circulating blood volume from the value of theexponential fit at the time of injection. For the experimental ICG traceshown in FIG. 4, the estimated cardiac output is 509 ml/min and thecirculating blood volume is 184 ml, in the expected range for a 3 Kgrabbit. This computer program is advantageous in that it improved theability to verify that the experimental measurements are proceeding asplanned or to correct without delay any measurement error orexperimental malfunction.

Indicator dosage. In this experiment is was found that a dose of about0.045 mg injected ICG was optimal in this animal to allow for detectionof an intense fluorescence dilution curve and at the same time rapidmetabolic disposal of the ICG. Further, with this small dose cardiacfunction measurements could be performed at about intervals of 4 minutesor less.

Detector placement. Defined fluorescence readings were obtained bypositioning the detection probe above the skin surface proximate to anartery or above tissue, such as the ear or the paw arterialized by localheating.

B. Calibration of Transcutaneous Indicator Intensity and CirculatingIndicator Concentration.

Calibration of the transcutaneous fluorescence intensity measured at thelevel of the animals' ear as a function of ICG concentration in bloodwas performed as follows. A high dose of ICG (1 mg) was injectedintravenously and equilibrated homogeneously with the animal's totalblood volume in about a one minute period. At equilibrium, the blood ICGconcentration resulting from this high dose is several times larger thanthe peak ICG concentration observed during the low dose ICG injections(0.045 mg) used to measure the cardiac output. In this way, acalibration curve was created that accommodated the full range of ICGconcentrations observed during the cardiac function measurements.

As the liver metabolizes ICG, the blood ICG concentration decreases backto 0 in about 20 minutes. During that time period, 5 to 8 blood samples(1.5 ml) were withdrawn from the femoral artery and placed in apre-calibrated blood cuvette. The fluorescence intensity of the blood inthe cuvette was converted to a measurement of concentration using theknown standard curve of fluorescence intensity versus ICG concentrationestablished for the cuvette. ICG fluorescence was measured at the levelof the ear at the exact time of the blood sample withdrawal. Because ICGis homogeneously equilibrated in the animal's blood volume, when theblood samples are withdrawn, the fluorescence intensity measured at thelevel of the ear corresponds directly to the ICG blood concentration atthe time of the measurement and therefore the ICG concentrationdetermined from the cuvette reading. As this example shows,transcutaneous ICG fluorescence is proportional to blood ICGconcentration such that a single blood withdrawal can suffice to findthe proportionality factor between the two quantities.

As shown in FIG. 5, the transcutaneous ear fluorescence intensity (in V)as a function of time (in s) after the high dose (1 mg) ICG injectionduring the calibration sequence. FIG. 5 shows the characteristic firstorder exponential decay of ICG in blood as the dye is being metabolized.FIG. 6 shows the ICG concentration (in mg/ml) as a function of the invivo fluorescence for the same example and the same time points. For therange of concentrations used in these studies, ICG concentration andtranscutaneous fluorescence were linearly related. The calibration linepasses through the origin of the axes since there is no measuredfluorescence when the ICG blood concentration is 0.

Thus, a simple proportionality factor exists between blood ICGconcentration and transcutaneous fluorescence. This feature of thefluorescence dilution technique measuring light emission is advantageousover the conventional dye dilution technique based on ICG absorptionwhich requires light absorption caused by ICG to be separated from lightabsorption by tissue and blood. After the proportionality factor isdetermined, ICG fluorescence dilution profiles can only then beconverted into concentration measurements for computation of the cardiacoutput using the indicator-dilution equation.

Results of cardiac output measurements. Calibrated cardiac outputreadings have been obtained in 8 animals (body wt: 3.0±0.2 Kg). Thefollowing table lists the values during baseline conditions. The valuesare presented as the mean±standard deviation of three consecutivemeasurements obtained within a 15 min period.

TABLE 1 Exp. Cardiac output (ml/min) 1 530 ± 15 2 500 ± 17 3 370 ± 12 4434 ± 16 5 481 ± 6 

The average for the five experiments (463 ml/min) is in order ofreported cardiac outputs (260-675 ml/min) measured with ultrasound orthermodilution techniques in anesthetized rabbits (Preckel et al. Effectof dantrolene in an in vivo and in vitro model of myocardial reperfusioninjury. Acta Anaesthesiol Scand, 44, 194-201, 2000. Fok et al. Oxygenconsumption by lungs with acute and chronic injury in a rabbit model.Intensive Care Med, 27, 1532-1538, 2001). Basal cardiac output variesgreatly with experimental conditionals such as type of anesthetic,duration and depth of anesthesia, leading to the wide range of valuesfound in the literature. In this example, the variability (standarddeviation/mean) of the calculated cardiac output with fluorescencedilution is ˜3% for any triplicate set of measurements which comparesfavorably with the reported variability for the thermodilution technique(˜5-10%).

C. Comparison of Measurements Obtained by Fluorescence Dilution CardiacOutput Method Via Transcutaneous Measurement and SubcutaneousMeasurement.

Experimental methodology. The experimental preparation described in thepreceding section (Example 2) includes two measurement sites for thefluorescence dilution traces: a transcutaneous site at the level of theear central bundle of blood vessels and the exposed femoral artery. Theear vasculature is arterialized by local heating. With this preparation,the cardiac output estimates obtained from the peripheral non-invasive(transcutaneous) measurement site were compared with estimates obtainedby interrogating a major artery.

The intensity of the fluorescence signal at the level of the exposedfemoral artery during the slow metabolic disappearance phase of theinjected ICG is compared to the calibrated ear fluorescence measurementto derive a calibration coefficient (arterial ICG fluorescence into ICGblood concentration). In this way cardiac output estimates expressed inml/min were derived from the two sites.

Results. FIG. 8 shows the time course of the cardiac output measurementsobtained from the ear site and from the exposed femoral artery in arepresentative experiment during control conditions (C), intense thenmild vagal stimulation (S,I and S,M), and post-stimulation hyperemia(H). Near-identical estimates of the cardiac output are obtained fromthe two sites during all phases of the study.

The relationship between cardiac output derived from measurement of thefluorescence dilution curve at the level of the skin surface (COskin, inml/min) and at the level of the exposed femoral artery (COfem, inml/min) was investigated. The linear relationships between the twomeasures are summarized in the table below:

TABLE 2 Regression Number Exp. Linear regression Coef. measurements 1COskin = 0.65(±0.11) * COfem + 0.81 22 145.0(±54.0) 2 COskin = 1.01(±0.06) * COfem + 0.96 27 2.0(±22.0) 3 COskin = 1.05(±0.14) * COfem −0.91 13 56.0(±54.0)

The two measures of fluorescence cardiac output are tightly correlated.In the last two experiments, the slope of the regression line is notstatistically different from 1.0 and the ordinate is not different from0.0 indicating that the two measurements are identical. Theseobservations suggest that fluorescence dilution cardiac output can bereliably measured transcutaneously and from a peripheral site ofmeasurement that has been arterialized by local application of heat.Attenuation of the excitation light and ICG fluorescence emission by theskin does not prevent the measurement of well-defined dye dilutiontraces that can be analyzed to derive the cardiac output.

While the specification describes particular embodiments of the presentinvention, those of ordinary skill can devise variations of the presentinvention without departing from the inventive concept.

D. Comparison of Measurements Obtained by Fluorescence Dilution CardiacOutput Method and Doppler Flow Velocity Technique.

Experimental methodology. The present method was compared with anultrasonic Doppler velocity probe method to record cardiac outputmeasurements. In this example the above procedure was modified in that,the animal's chest was opened with a median incision of the sternum anda 6 mm 20 MHz Doppler velocity probe was gently passed around theascending aorta and tightened into a loop that fits snuggly around theaorta.

For detection of the fluorescent detection of the indicator, twoillumination+detection fiber optic probes were used: one probe wasplaced on or above the ear middle vessel bundle and the other probe wasplaced in proximity to the dissected left femoral artery. Local heatingto 42 degrees centigrade arterialized the ear vasculature.

In this example, two maneuvers were used to change the cardiac outputfrom its control level: vagal stimulation, which reduces the cardiacoutput, and saline infusion, which increases the circulating volume andcardiac output. The right vagal nerve was dissected to position astimulating electrode. Stimulation of the distal vagus results in a moreor less intense decrease of the heart rate that depends on thestimulation frequency and voltage (1 ms pulses, 3 to 6 V, 10 to 30 Hz).The cardiac output and aortic flow velocity also decrease during vagalstimulation even though less markedly than the heart rate decreasesbecause the stroke volume increases. Saline infusion at a rate of 15-20ml/min markedly increases the cardiac output. FIG. 7 shows the timecourse of the cardiac output and aortic velocity measurements in oneexperiment including control conditions (C), intense then mild vagalstimulation (S,I and S,M), and saline infusion (I).

Results. There is consistent tracking of the Doppler aortic velocity bythe fluorescence dilution cardiac output measurement. The relationshipbetween fluorescence dilution cardiac output and aortic Doppler flowvelocity was investigated in four rabbits. The linear relationshipsbetween fluorescence dilution cardiac output (CO, in ml/min) and aorticflow velocity signal (VAor, not calibrated, in Volts) are summarized inthe table:

TABLE 3 Regression Exp. Linear regression Coef. Measurements 1 CO =789(±123) * VAor + 0.79 27 166(±34) 2 CO = 607(±62) * VAor + 50(±32)0.90 24 3 CO = 614(±64) * VAor − 45(±38) 0.90 27 4 CO = 654(±41) * VAor− 3(±29) 0.97 18

This data indicates that the fluorescence dilution cardiac output ishighly correlated with aortic flow velocity as indicated by the elevatedregression coefficient (≧0.9 in 3 experiments). Further, the slopes ofthe linear regression lines between fluorescence dilution cardiac outputand aortic flow velocity are similar and statistically not different inthe four studies. This suggests a constant relationship between the twovariables across experiments. The ordinates of regression lines are notdifferent from 0 in the last three experimental studies, which suggestsabsence of bias between the two measures of aortic flow.

The results above establish that fluorescence dilution cardiac outputmeasured transcutaneously tracks the Doppler flow velocity measured inthe ascending aorta.

EXAMPLE 3

Comparison with Thermodilution Method

Experimental methodology. Other experiments were performed in NewZealand White rabbits using the methodology described for the precedingexample 2. In addition, a 4 F thermodilution balloon catheter wasinserted into the right femoral vein and advanced until the thermistorreached the main pulmonary artery. Correct placement of the catheter tipwas verified visually through the thoracotomy. The catheter wasconnected to a cardiac output computer to measure the thermodilutioncardiac output. Cardiac output measurements were obtained with thepresent method (CO_(ICG)) and the comparison thermodilution method(CO_(TD)) during baseline conditions, reduced flow conditions resultingfrom vagal stimulation, and increased flow conditions resulting fromblood volume expansion with saline.

Results. Average values of CO_(ICG) and CO_(TD) measured in baselineconditions in the 10 animals were 412 (±13) ml/min and 366 (±11) ml/min,respectively, in the expected range for anesthetized rabbits. In eachanimal, CO_(ICG) was linearly related to CO_(TD) as shown on thefollowing table 4. The slope of the regression line (range: 0.74-1.25)was not different from 1.0 in 8 studies. In the combined data from all10 studies the linear relationship between CO_(ICG) and CO_(TD) had aslope (0.95±0.03) not different from 1.0 and an ordinate (77±10 ml/min)that was slightly >0.

TABLE 4 Experiment EQUATION N R 1 CO_(ICG) = 0.94^(†)(±0.08) CO_(TD) +84(±23) 21 0.94 2 CO_(ICG) = 1.25^(†)(±0.17) CO_(TD) − 0*(±39) 17 0.88 3CO_(ICG) = 0.74(±0.11) CO_(TD) + 122(±26) 20 0.85 4 CO_(ICG) =0.90^(†)(±0.05) CO_(TD) + 98(±15) 11 0.99 5 CO_(ICG) = 1.08^(†)(±0.11)CO_(TD) + 84(±47) 14 0.94 6 CO_(ICG) = 1.07^(†)(±0.09) CO_(TD) +16*(±33) 14 0.96 7 CO_(ICG) = 1.15(±0.06) CO_(TD) + 29*(±25) 12 0.99 8CO_(ICG) = 0.82^(†)(±0.09) CO_(TD) + 83(±37) 12 0.94 9 CO_(ICG) =0.88^(†)(±0.12) CO_(TD) + 98*(±62) 16 0.89 10 CO_(ICG) = 1.05^(†)(±0.08)CO_(TD) − 20*(±33) 15 0.97 All CO_(ICG) = 0.95^(†)(±0.03) CO_(TD) +74(±10) 152 0.94

These studies further established that cardiac output CO_(ICG) measuredwith the present method is linearly related to thermodilution cardiacoutput CO_(TD). The slope of the regression line between these variableswas near 1.0 for most experiments, as well as for the grouped data fromall experiments.

EXAMPLE 4

A. Noninvasive Calibration

One embodiment of the calibration system includes a method to determinenon-invasively transcutaneously the concentration of a fluorescentindicator injected in the bloodstream by measuring the intensity of thefluorescence light emitted by the indicator when illuminated by a lightsource in or near the skin and the intensity of the light from thatsource reflected by or transmitted through the illuminated skin site.

In the pulse dye densitometer (Cardiac output and circulating bloodvolume analysis by pulse dye densitometry. Iijima T. et al. Journal ofClinical Monitoring, 13, 81-89, 1997, incorporated herein in itsentirety by reference), light absorption is measured at two wavelengths:805 nm where ICG absorption is near maximum and 890 nm where ICGabsorption is very small. Assuming at first that tissue absorption oflight is only due to blood hemoglobin and ICG, the ratio C_(ICG)/C_(Hb)can be expressed as a function of the ratio Ψ of the optical densitiesmeasured at 805 nm and 890 nm,

${C_{ICG}/C_{Hb}} = \frac{E_{{Hb},805} - {\Psi\; E_{{Hb},890}}}{{\Psi\; E_{{ICG},890}} - E_{{ICG},805}}$

where E represents the absorption coefficient from Beer's Law. Thelatter is expressed as I_(x)=I₀e^(−E.C.x) with C=concentration,E=absorption coefficient, x=pathlength in substance. Note that if weassume that E_(ICG),890=0, the ratio of the concentrationsC_(ICG)/C_(Hb) is linearly related to the ratio of the optical densitiesmeasured at two wavelengths.

Taking into account scattering and absorption by other material besideICG and Hb, the developers of the pulse dye densitometer establishedthat the ratio of the optical density changes between before and afterICG administration at 805 nm and 890 nm could be expressed as a functionof the ratio C_(ICG)/C_(Hb).

ICG fluorescence is proportional to the absorption of light by ICG atthe wavelength of excitation (805 nm in the model above or 784 nm in ourstudies). Therefore, we hypothesized that the ratio C_(ICG)/C_(Hb) canbe derived from the ratio of the change in light signal measured at thewavelength of emission (related to ICG fluorescence) to the light signalmeasured at the wavelength of excitation (related to ICG and Hbabsorption).

We considered a model of light propagation in tissue, which at firstassumed that only hemoglobin and ICG were absorbers (See Table 5 below).The absorption coefficients of ICG and Hb were derived from theliterature and considered to be independent of wavelength. We then addeda dependence of the absorption coefficients on wavelength and tissueabsorption in the model to investigate the effect of these factors.

TABLE 5 1-D model of light propagation and fluorescence generation

The following data and assumptions were applied to the model of Table 5:

-   -   μ_(a,ICG)=38.1 μl.μg⁻¹.mm⁻¹ for wavelength λ=784 nm;    -   μ_(a,HbO2)˜μ_(a,Hb)=0.0026 μl.μg⁻¹.mm⁻¹ for wavelength λ=784 nm;    -   Initially, we assume that the absorption coefficients have the        same values at 830 nm (fluorescence) and at 784 nm (incident        excitation light);    -   C_(Hb)=12−18 g.dl⁻¹=120−180 μg/μl in blood;    -   C_(ICG) max=0.005 μg/μl in blood;    -   Tissue assumed to contain 10% blood;    -   Quantum yield of ICG fluorescence=0.04;    -   Transmission calculated through 40 mm tissue in 0.02 mm        increment.

We modeled transmission and fluorescence signals at 784 nm and 830 nmfor different ICG concentrations and hemoglobin contents when absorptioncoefficients are the same, and the results are illustrated in the graphsof FIGS. 11A-11D. For this simple model, the transmitted excitationlight decreases nonlinearly as a function of ICG concentration in themodel and the curve varies with the hemoglobin content (see FIG. 11A).Also the emergent fluorescence light increases nonlinearly with ICGconcentration (inner filter effect), and the curve varies withhemoglobin content (see FIG. 11B). Thus, the fluorescence signal variesmarkedly if there is more or less absorption by blood in the tissue.

However, the ratio (emergent fluorescence light/transmitted excitationlight) is proportional to the ICG concentration and independent of thehemoglobin content of the tissue (see FIG. 11C). Therefore, by measuringthe ratio and if the relationship is known, the ICG concentration can beestimated. Also, the ratio (emergent fluorescence light y/transmittedexcitation light x) is proportional to the ratio (ICG concentration/Hbconcentration) but in this case the slope varies with the hemoglobincontent of the tissue (see FIG. 11D). In an alternative embodiment ofthe calibration system, the concentration of Hb may be obtained from ablood sample, and this concentration value can be used to determine theratio of ICG value to Hb value, which can then be used with the ratio oftransmitted excitation light to fluorescence light to determine theconcentration of ICG for calibration.

We also modeled transmission and fluorescence signals and at 784 nm and830 nm for different ICG concentrations and hemoglobin contents whenabsorption coefficients are the different and an additional absorber isincluded, and the results are illustrated in the graphs of FIGS.12A-12D.

Absorption by ICG is actually slightly more elevated at 784 nm(excitation) than it is at 830 nm (fluorescence peak). In contrastoxy-hemoglobin absorption is less at 784 nm (excitation) than it is at830 nm. In addition to blood hemoglobin and ICG, bloodless tissueabsorbs to a certain extent. We determined various values from theliterature:

μ_(a,ICG)=38.1 μl.μg⁻¹.mm⁻¹ for wavelength λ=784 nm;

μ_(a,HbO2)˜μ_(a,Hb)=0.0026 μl.μg⁻¹.mm⁻¹ for wavelength λ=784 nm;

μ_(a,ICG)=34.1 μl.μg⁻¹.mm⁻¹ for wavelength λ=830 nm;

μ_(a,HbO2)˜μ_(a,Hb)=0.0035 μl.μg⁻¹.mm⁻¹ for wavelength λ=830 nm

μ_(a,tissue)=0.1.mm⁻¹ independent of wavelength in the range 784-830 nm;

C_(Hb)=12-18 g.dl⁻¹=120-180 μg/μl in blood;

C_(ICG) max=0.005 μg/μl in blood;

Tissue assumed to contain 10% blood;

Quantum yield of ICG fluorescence=0.04;

Transmission calculated through 40 mm tissue in 0.02 mm increment.

For this more complete model, the magnitude of the transmittedexcitation light and emergent fluorescent lights are markedly decreasedwhen compared to the first model primarily because of the absorption bybloodless tissue. Both signals follow the pattern found for the simplemodel. In particular, the emergent fluorescence light increasesnonlinearly with ICG concentration (inner filter effect) and the curvevaries with hemoglobin content.

As before the ratio (emergent fluorescence light/transmitted excitationlight) is proportional to the ICG concentration (See FIG. 12C). Whilethe slope is dependent on the hemoglobin content, there are only smalldifferences between the four levels of hemoglobin considered. Thissuggests that by measuring the ratio of the fluorescence/transmittedlight, the ICG concentration can be estimated once the linearrelationship is determined and possibly including a factor that accountsfor the hemoglobin content.

While these models do not consider tissue scattering, the latter isoften assumed to increase the pathlength of light in tissue by a fixedproportionality factor: the pathlength factor (about 3.6 for humanforearm, see Measurement of hemoglobin flow and blood flow bynear-infrared spectroscopy. Edwards A. D. et al.—J. Appl. Physiol. 75,1884-1889, 1993, the entire contents of which are incorporated herein byreference). This suggests that the model analysis above would likelyremain valid even in the presence of scattering.

Other biocompatible fluorescent dyes such as fluorescein and rhodaminewould also be suitable in the noninvasive calibration of the example 4above. Fluorescein in blood plasma has a peak fluorescence of about518±10 nm with an optimal excitation wavelength of about 488 nm(Hollins, supra; Dorshow, supra). Rhodamine in blood plasma has a peakfluorescence of about 640±10 nm with an optimal excitation wavelength ofabout 510 nm.

The previous description of the disclosed embodiments is provided toenable any person skilled in the art to make or use the cardiac outputmonitor devices, methods and systems. Various modifications to theseembodiments will be readily apparent to those skilled in the art, andthe generic principles defined herein may be applied to otherembodiments without departing from the spirit or scope of the devices,methods and systems described herein. Thus, the cardiac output, devices,methods and systems are not intended to be limited to the embodimentsshown herein but are to be accorded the widest scope consistent with theprinciples and novel features disclosed herein.

1. A method of determining concentration of a fluorescent indicator thatis indocyanine green (ICG) having a concentration C_(ICG) within thecardiovascular system of a subject comprising: a. administering to thecardiovascular system a detectable amount of the indicator thatfluoresces when illuminated; b. illuminating a portion of thecardiovascular system with an excitation light of a first wavelength andcausing the administered indicator to emit an emergent fluorescencelight of a second wave length; c. detecting the intensity (x) of theexcitation light that is reflected by or transmitted through a portionof the cardiovascular system; d. detecting the intensity (y) of theemergent fluorescence light; e. determining the ratio (y)/(x); f.determining the concentration (C_(ICG)) of the fluorescence indicator inthe cardiovascular system based on the determined ratio (y)/(x), whereinthe concentration of the fluorescence indicator is proportional to theratio; and g. determining a ratio C_(ICG)/C_(Hb) of indocyanine greenconcentration (C_(ICG)) to the blood hemoglobin concentration (C_(Hb))wherein the ratio C_(ICG)/C_(Hb) is proportional to the change of theratio (y)/(x).
 2. The method of claim 1, wherein the first wavelength isselected from a range of about 488 nm to about 1000 nm.
 3. The method ofclaim 2, wherein the indicator is any one of fluorescein or rhodaminecapable of emitting the second wavelength of light in a range from about500 nm to about 900 nm.
 4. A system for determining concentration of afluorescent indicator that is indocyanine green (ICG) having aconcentration C_(ICG) within the cardiovascular system of a subjectcomprising: a. an illumination source configured to be positionedproximately to at least one blood vessel of the cardiovascular system toprovide a first wavelength of light to excite an indicator within thecardiovascular system to cause the indicator to emit a second wavelengthof light; b. at least one photodetector configured to be positionedproximate to the cardiovascular system of the subject and configured todetect information regarding the intensity (x) of the excitation lightthat is reflected by or transmitted through a portion of thecardiovascular system and to detect information regarding the intensity(y) of the emergent fluorescence light; and c. a computing systemconfigured: to receive the detected intensity information from thephotodetector; ii. to determine the concentration (C_(ICG)) of theindicator, wherein (C_(ICG)) is proportional to the ratio (y)/(x); andiii. to determine a concentration ratio C_(ICG)/C_(Hb) of indocyaninegreen concentration (C_(ICG)) to the blood hemoglobin concentration(C_(Hb)), wherein the ratio C_(ICG)/C_(Hb) is proportional to the changeof the ratio (y)/(x).
 5. The system of claim 4, wherein thephotodetector is configured for placement in at least one of atransdermal detection area, a subdermal detection area, a perivasculardetection area or an endovascular detection area.
 6. The system of claim5 further comprising at least one fiber optic probe operably connectedto the illumination source and configured to guide the first wavelengthof light from the illumination source to the detection area.
 7. Thesystem of claim 5 further comprising at least one fiber optic probeoperably connected to the photodetector configured to guide the secondwavelength of light from the detection area to the photodetector.
 8. Thesystem of claim 4, wherein the first wavelength is selected from a rangeof about 488 nm to about 1000 nm.
 9. The system of claim 4, wherein theindicator is any one of fluorescein or rhodamine capable of emitting thesecond wavelength of light in a range from about 500 nm to about 900 nm.